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Daniel M. Lipinski, Mandeep Singh, Peter Charbel Issa, Alun R. Barnard, Robert E. MacLaren; CNTF Sustains Cone Photoreceptors in a Novel In Vitro Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5457.
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© ARVO (1962-2015); The Authors (2016-present)
The decline of central vision in the late stages of retinitis pigmentosa (RP) is due to the loss of cone cells secondary to advanced rod degeneration. The attenuation of cone photoreceptor degeneration by the delivery of neurotrophic factors has the potential to yield a widely applicable treatment for rod-cone dystrophies. Here we evaluated the neuroprotective effects of ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) using a novel in vitro model of RP that allows longitudinal quantification of green fluorescent protein (GFP)-expressing cone photoreceptors in organotypic culture.
Retinal explants were harvested from 10 week old rhodopsin knockout mice which had been crossed with OPN1.gfp reporter mice, so that GFP positive cones could be identified against the background of a rod-specific degeneration. An organotypic culture system was used to maintain the explants in viable condition. Neurotrophic factors (200ng/ml, 100ng/ml or 50ng/ml; n=5 explants per group) were administered daily to treatment groups. Fluorescence microscopy was performed on days 1, 3, 5, 7, 9 and 12 to document the number of GFP-expressing cones with statistical comparisons performed using the Tukey-Kramer multiple comparison test.
Daily administration of 200ng/ml CNTF led to increased cone survival compared to negative controls at all time points (p=<0.001). The observed effect was dose dependent, where 100ng/ml CNTF reduced cone loss at day 5 (p=<0.05) through day 12 (p=<0.001). Negative controls showed a significant decline in cone photoreceptor numbers between day 3 and day 12 (p=<0.001). 200ng/ml GDNF showed significant protection against cone loss at later time points (day 9, p=<0.05; day 12 p=<0.001) but was much less effective than CNTF at all doses.
Cone survival in this in vitro model of RP was significantly enhanced by the administration of CNTF and GDNF, which supports their potential role as therapeutic agents to attenuate cone loss in RP. The organotypic culture system described here is a robust method to screen neuro-protective compounds in vitro prior to their potential in vivo delivery via gene therapy.
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